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Zygote
2015 Jun 01;233:426-46. doi: 10.1017/S0967199414000033.
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Ca²⁺ influx-linked protein kinase C activity regulates the β-catenin localization, micromere induction signalling and the oral-aboral axis formation in early sea urchin embryos.
Yazaki I, Tsurugaya T, Santella L, Chun JT, Amore G, Kusunoki S, Asada A, Togo T, Akasaka K.
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Sea urchin embryos initiate cell specifications at the 16-cell stage by forming the mesomeres, macromeres and micromeres according to the relative position of the cells in the animal-vegetal axis. The most vegetal cells, micromeres, autonomously differentiate into skeletons and induce the neighbouring macromere cells to become mesoendoderm in the β-catenin-dependent Wnt8 signalling pathway. Although the underlying molecular mechanism for this progression is largely unknown, we have previously reported that the initial events might be triggered by the Ca2+ influxes through the egg-originated L-type Ca2+ channels distributed asymmetrically along the animal-vegetal axis and through the stretch-dependent Ca2+channels expressed specifically in the micromere at the 4th cleavage. In this communication, we have examined whether one of the earliest Ca2+ targets, protein kinase C (PKC), plays a role in cell specification upstream of β-catenin. To this end, we surveyed the expression pattern of β-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I). Unlike previous knowledge, we have found that the initial nuclear entrance of β-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage. Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of β-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.
Figure 1. Inhibition of PKC interferes with the normal distribution of actin cytoskeleton in blastomeres. (A) Preparation of the HpPKC inhibitor (HpPKC-I). The amino acid sequence of Hemicentrotus pulcherrimus PKC was aligned with Lytechinus pictus (LpPKC) and human PKC (HuPKC) (1–68) to define the pseudosubstrate region (underlined nine residues). Asterisks denote identical amino acid residues. The synthetic peptides of the pseudosubstrate were myristoylated to use as HpPKC-I. (B) Effects of PKC on actin distribution. F-actin was visualized with Alexa Fluor 488-conjugated phalloidin; 8 nM PMA was added to the embryos (P. lividus) for 20 min starting from 7 min before the 4th division; 5 μM of HpPKC-I was added 20 min before the 4th division. Optical (upper) and fluorescent images (lower) were taken from the same confocal plane. In either case, cell specifications were modified, but cell division progressed normally.
Figure 3. Distribution of Hpβ-catenin during mitosis. Embryonic cells at various mitotic stages (H. pulcherrimus) were stained with Hpβ-catenin antibody (green) and PI (red). Cells marked with asterisks in each embryo of (A), (B), (C) and (D) respectively were enlarged in the right-side panel (a, b, c, d). (A) At the 56-cell stage, most macromere and all mesomere derivatives shown were in prophase. (B) Cells at metaphase and anaphase in the 28-cell stage embryo. (C) Cells at telophase in the 28-cell stage embryo from the ventral view. Macromeres have just divided to be in telophase, showing an irregular, chambered structure in the process of reconstruction of the daughter nuclei. (D) A ventral view of 28-cell stage embryo. Macromeres were at interphase.
Figure 4. Localization of the nuclear β-catenin in the 16-cell to 56-cell stage embryos. (A1–E1) double staining of propidium iodide (PI) (red) and β-catenin antibody (green). (A2–E2) images of β-catenin staining only. (A3–E3) Drawing of (A1–E1) images to illustrate the nuclei and the contour lines of blastomeres. Abbreviations: mic; micromeres, Mac; macromeres, meso; mesomeres. (A) H. pulcherrimus embryo at the 16-cell stage: all cells were at interphase. Nuclear β-catenin was preferentially present in Mac. (B) Late 16-cell stage: mic remained at the interphase, but Mac progressed to anaphase. No β-catenin was found in mic nuclei. (C) Early 28-cell stage: Mac divided to the telophase, and mic formed chromosomal plate of metaphase. (D) At the 28-cell stage: mic at metaphase or anaphase. Mac and meso derivatives were at interphase. Nuclear β-catenin was detected only in Mac derivatives. (E) At the 56-cell stage: s-mic, small micromeres; l-mic, large micromeres. Veg1 and Veg2 are the macromere derivatives locating at the animal side (an) or next to l-mic, respectively. All cells shown were at interphase, and nuclear β-catenin was detected in every cell except for ‘an (animal side)’ cells. Abbreviations: An, animal side; Vg, vegetal side.
Figure 5. LiCl, PKC activator and PKC inhibitors affect the distribution of β-catenin. Localization of β-catenin (green) and nuclear staining (red) in the H. pulcherrimus embryos at the 16-cell, 28-cell and 56-cell stages. (A) Control embryos. (B) Embryos treated with 40 mM LiCl starting from the 4-cell stage. (C) Embryos exposed to PKC activator (8 nM PMA for 20 min starting from 5 min before the 4th cleavage). (D, E) Embryos exposed to 5 μM HpPKC-I starting from 20 min before the 4th division (16-cell stage). (F) Embryos exposed to 400 nM Gö6976, a calcium-dependent PKC inhibitor, for the same period as HpPKC-I. Abbreviations: An, animal side; Vg, vegetal side; mic; micromeres, Mac; macromeres, meso; mesomeres.
Figure 6. Inhibition of Ca2+infux and of PKC activity delayed gastrulation, but not PMC ingression. (A) P. lividus embryos were treated with 25 μM GdCl3 for 50 min starting from 15 min before the 2nd, 4th, 5th and 7th cleavages at room temperature. Cell cycles went on every 30–35 min. Control and GdCl3-treated embryos were both fixed at the same time, and the percentage of PMC-ingressed embryos or gastrulation-initiated embryos were calculated from circa 100 embryos from two or three independent experiments, respectively. (B, C) H. pulcherrimus embryos were treated with HpPKC-I at 6 μM (B) or 5 μM (C) during the period indicated by the horizontal bars. Gastrulation levels were estimated only from the side-viewed embryos. Embryos were cultured at 15°C (B), at 18°C (C). The cell cycles of these experiments were 45–48 min (B) and 39 min (C).
Figure 7. HpPKC inhibitor caused similar morphological changes on embryos of H. pulcherrimus and P. lividus. Embryos were treated with 5 μM HpPKC-I from 20 min before the 4th cleavage to 20 min after the 6th cleavage. This period was about 120 min for H. pulcherrimus culturing at 18°C and 100 min for P. lividus at 23°C. Control and HpPKC-I-treated embryos. (A) H. pulcherrimus embryos; early blastulae at 6 h post fertilization (p.f.), gastrulae at 22 h, and plutei at 52 h (B) P. lividus; morulae at 5 h, gastrulae at 21 h, and plutei at 47 h. (C) Names of the skeletal parts of the pluteus.
Figure 8. Morphological changes of H. pulcherrimus embryos treated with HpPKC inhibitor before the 8-cell stage. (A) Control embryos. (B) Embryos treated with 5 μM HpPKC-I starting from the 4-cell stage (20 min before the 3rd cleavage) to the end of 8-cell stage. (C) Embryos treated with 5 μM HpPKC starting from 20 min before the 4th cleavage to the end of 16-cell stage. Embryos were cultured at 18°C. The hand-drawn delineations on the right panels represent the embryos marked with asterisks in (A–C). Oral ectoderm area and aboral area were indicated by white arrowheads and black arrows, respectively.
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